Protein compartmentation within cells consists of protein targeting to specific organelles and transport across their membranes, followed by membrane vesicular traffic among the organelles. We study protein transport across membranes through the "Sec" and "Tat" preprotein translocases of E. coli. As we study each, we hope to understand how one transports unfolded proteins and the other only folded proteins. We explore vesicle traffic by studying the homotypic (self) fusion of vacuoles from S. cerevisiae. Each of these two studies, while exploiting the excellent genetics of the organisms, takes a fundamentally enzymologic approach of identifying and purifying each of the necessary proteins, lipids, and cofactors and, with these pure elements, reconstituting the biological reaction. We have purified and studied the catalytic cycle of the E. coil "sec" preprotein translocase, and we are now studying its subunit structure and mechanism as we embark on the isolation of the Tat translocase. Vacuole fusion has been recapitulated in vitro with isolated vacuoles and a few pure soluble components, but relies on a complex array of membrane-bound proteins, 17 of which are identified and under study. Genetic tests suggest that there are approximately 50 more proteins involved in this reaction, and genetic and biochemical methods are described for their identification and isolation. The same proteins which catalyze protein secretion into the blood or transmission at the neural synapse are also responsible for vacuole fusion; thus, basic mechanisms discovered in this work will be of broad importance to biology and medicine.